UNIVERSITI PUTRA MALAYSIA PLASMID PROFILING AND …psasir.upm.edu.my/id/eprint/8372/1/FSMB_1997_1_A.pdf · 2013-05-27 · A schematic diagram of isolation and identification of ...

UNIVERSITI PUTRA MALAYSIA

PLASMID PROFILING AND ANTIBIOTIC RESISTANCE OF ESCHERICHIA COLI AND ESCHERICHIA COLI 0157 STRAINS

SAHILAH ABD. MUTALIB

FSMB 1997 1

PLASMID PROFILING AND ANTIBIOTIC RESISTANCE OF ESCHERICHIA COLI AND ESCHERICHIA COLI 0157 STRAINS

By

SAHILAH ABO. MUTALIB

Dissertation Submitted tn Fulfilment of the Requirements for the Degree of Master of Science in the Fa"culty of

Food Science and Biotechnology,

Universitl Putra Malaysia.

MAY 1 997

ACKNOWLEDGEMENTS

The author wishes to thank her chairman Dr. Son Radu for his dedicated

efforts, guidance, patience, supervision and constant encouragement throughout the

research.

The author also extends her gratitude and appreciation to her member of

supervisory committee, Associate Professor Dr. Gulam Rusul Rahmat Ali for his

strong support, supervision, encouragement and advise given. Sincere gratitude is

also extended to her members of supervisory committee, Dr. Raha Abdul Rahim for

her moral support, advise and encouragement throughout the research.

Last but not least, the author wishes to thank her beloved husband Dr.

Ahmad Azuhairi, Abdullah and family, Wani, parents, in-laws and all her friends

(Ahmad Zainuri, Kak Maimunah, Kak Endang Purwati Ningseh, Lew Wang Shiang,

Loke Siew Fung and Dayang Fadzelina) for their constant moral support.

ii

TABLE OF CONTENTS

ACKNOWLEDGEMENTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . LIST OF T ABLES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LIST OF FIGURES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. .

LIST OF PLATES . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

LIST OF ABBREVIATIONS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ABSTRACT . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

ABSTRAK ........................................................................................... .

CHAPTER

1 INTRODUCTION . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '"

Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

2 LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Escherichia coli ............................................................ .

Taxonomy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Habitat . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Isolation and Identification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Serological Classification . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Escherichia coli in Diarrheal Diseases . . . . . . . . . . . . . . . . . . . . Escherichia coli 0 157:H7 .......................................... . .

Biochemical Characteristic for Identification of Escherichia coli 0157 :H7 ....................... ......... ..

Epidemiology and Transmission . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Syndromes Caused by Escherichia coli 0157:H7 .. .

Pathogenicity . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Use of Antibiotic in Animals . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Bacterial Resistance to Antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Genetic Basis of Bacterial Resistance to Antibiotic . . . . .

Plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

R plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Plasmid in Escherichia coli and Escherichia coli 0157 :H7 ...................................... .

Transposons . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Transfer Mechanism . . . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . Conjugation . . . . . . . . . . . . . . . . . . . . ........ . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Transduction . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Transformation . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

iii

Page

11 VI

V11 IX X

Xl xiii

1 4

5 5 5 6 7

10 12 17

17 18 19 20 23 24 26 27 31

38 39 48 48 49 50

3

4

5

Mechanism of Antibiotic Resistance Mediated by R Plasmids . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

ISOLATION AND IDENTIFICATION OF ESCHERICHIA COLI AND ESCHERICHIA COLI 0157 . . . . . .... . . . . . . . ... . . . . ..... . . . . . . . ... . . .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . .. Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Samples Collection ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . Isolation and Identification of Escherichia coli and Escherichia coli 0 1 57 from Imported Frozen Beef . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Biochemical Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Results ........................... .............................................. .

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Conclusions .. . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .. . . . .... . . . .. . . . . . . . . . . . . . . . . . .

PLASMID PROFILING AND ANTIBIOTIC SUSCEPTIBILITY TESTS . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Methods . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Plasmid Isolation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Agarose Gel Electrophoresis . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Determination of Molecular Weight of Plasmid . . . . . . McFarland Turbidity Standard . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Antibiotic Susceptibility Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Interpretation of �one of Inhibition . . . . . . . . . . . . . . . . . . . . . . . .

Results . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Conclusions . . . . . . . . . . . . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

CONJUGA TION AND TRANSDUCTION/ TRANSFORMATlqN . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . . . .

Introduction . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... . . .

Methods . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

C .

t' onJuga Ion .. . . . . . . .... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Transduction/Transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Results . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Discussion . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Conclusions . . . . . . . . . . . . . . . . .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . .

iv

50

53 53 54 54

54 57 58 63 64

66 66 67 67 68 69 69 70 70 72 89 91

92 92 93 93 94 95 99

1 0 1

6 GENERAL DISCUSSION AND CONCLUSION . . . . . . .. . . . . . . . . 102

REFERENCES . . . . .... . ..... . ..... . . . . . . . . . . . . . . . .. . ... . . . . . . ...... . . .... . ... ... . ........ .. ... .. . 105 APPENDIX ..... ....... . . ......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 8 VITA .................................................................................................. 1 27 LIST OF PUBLICATIONS. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128

v

LIST OF TABLES

Table Page

1 The biochemical reactions of Escherichia coli Edwards and Ewing (1986) ....................... ..................................... 9

2 The K antigen of Escherichia coli.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 11

3 Comparison of stable toxin (ST) and labile toxin (L T) of Escherichia coli ............ .... ............ .... ............... . .. . . ... . . .. . ... 14

4 Mechanism of resistance to antibiotics . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

5 Some well-studied transposable elements . . . . . . . . . . . . . . . . . . . . . . . 45

6 Escherichia coli strains isolated from four different in Selangor and Federal Territory with their biochemical and analysis from recommended methods of Edwards and Ewing (1972) . . . . .. . . . . . . . .. . . . . . . .. ..... .. ....... .. . . . . . 59

7 Characterisation and identification of Escherichia coli 0157 .... . . . . . . . . ....... . . . .. . . .. . .... . . .. . . ....... .... ... 60

8 Sizes (in megadaltons) of V 517 size reference plasmids used to estimate the molecular weight of ccc plasmids in Escherichia coli 0157 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

9 Antibiotic susceptibility and plasmid patterns among Escherichia coli isolates from imported frozen beef.. . . . . 73

10 Antibiotic susceptibility of Escherichia coli isolates with and without plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . 77

11 Antibiotic susceptibility and plasmid patterns among Escherichia coli 0157 isolates from imported frozen beef ................................................................................. 84

12 Antibiotic susceptibility of Escherichia coli 0157 isolates with and without plasmid DNA . . . . . . . . . . . . . . . . . . . . . . . . 88

13 Frequency of transfer of resistance markers and phenotypes of Escherichia coli 0157 transconjugants . . . 96

14 Frequency of Escherichia coli 0157 transduction/transformation . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 97

vi

15 Antibiotic resistance and plasmid sizes of Escherichia coli 0157 isolates and their transconjugants ............................................................... .

vii

97

Figure

1

2

3

4

5

6

7

8

LIST OF FIGURES

Circular and linear forms of plasmid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Heteroduplexes formed between related F-like plasmids ........................................................................... .

Positions of gene coding for drug-resistance within the r-determinant region ofRl drdl9 ............................. ...... .

Simplified common features of the transposable elements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Structure of transposon Tn3 . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

Replicative and conservative transposition . . . . . . . . . . . . . . . . . . . . .

A schematic diagram of isolation and identification of Escherichia coli and Escherichia coli 0 1 57 from imported frozen beef . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..

Graph of electrophoretic mobility (m) against log of molecular weight (megadalton) for V5 1 7 ..................... .

viii

Page

28

36

37

42

44

46

56

70

LIST OF PLATES

Plate Page

1 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0 1 57 isolates .............................................. . 79

2 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0 1 57 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 80

3 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0 1 57 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 8 1

4 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0 1 57 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 82

5 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0157 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 83

6 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0157 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

7 Agarose gel (0.7%) electrophoresis of plasmid DNA Escherichia coli 0 1 57 isolates . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 87

8 Agarose gel (0.7%) electrophoresis of trans con jug ants plasmid DNA Escherichia coli 0 1 57 isolates . . . . . . . . . . . . . . . . . . . . . . . 98

ix

BCIG ccc CO2 CT-SMAC DNA EDTA EMBA g HCI IMViC KH2P04 mm MUG ml Na2HP04 NaOH % oc SDS SMAC TBE UV �l w/v

LIST OF ABBREVIATIONS

5 bromo-4-chloro-3-indolxyl-I3-D-glucuronic Covalently close circular Carbon dioxide Cefizime tellurite sorbitol MacConkey agar Deoxyribonucleic acid Ethylenediaminetetra-acetic acid Eosin methylene blue agar gram Hydrocloric acid Indole methyl red Voges-Prokauer citrate Potassium phosphate minute 4-methylumbelliferone glucuronide Milliliter di-sodium phosphate Natrium hydroxide Percentage Open circular Sodium dodecyl sulphate Sorbitol MacConkey agar Tris-Boric-EDTA Ultra violet Microliter Weight per volume

x

Abstract of thesis submitted to the Senate ofUniversiti Putra Malaysia in fulfilment of the requirements for the degree of Master of Science.

PLASMID PROFILING AND ANTIBIOTIC RESISTANCE OF ESCHERICHIA COLI AND ESCHERICHIA COLI 0 1 57 STRAINS

By

SAHILAH ABO. MUTALIB

MAY 1 997

Chairman: Dr. Son Radu.

Faculty : Food Science and Biotechnology.

Enterohaemorrhagic Escherichia coli 01 57:H7 has received considerable

amount of attention because of its implication in sporadic outbreaks of hemorrhagic

colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic purpura. The

incidence of E. coli 01 57:H7 have been reported in various parts of the world,

however, its incidence in MalaysiJl is relatively unknown. From March to June

1 996, 25 beef samples were obtained from 4 supermarkets in Selangcr and Federal

Territory, Malaysia. Twenty five Po of beef samples were enriched in 225 ml of EC

proth containing novobiocin (20 Qlgll) for four hours at 37°C and were plated on

MacConkey agar. Nineteen to tift) lactose fermenting colonies per sample (n=109 1 ) •

were transferred onto Eosin Meth!,lene Blue Agar, Sorbitol MacCoqkey Agar and

Cefizime Tellurite Sorbitol MacCe:nkey Agar. Sixty five of 112 metallic sheen and

sorbitol negative colonies were PQsitive when tested for 0 1 57 antigen using latex

agglutination test kit of Oxoid and Serobact. Nineteen (76%) samples were positive

xi

for E. coli 0 1 57:H7. The 65 strains of E. coli were tested for susceptibility against

20 antimicrobial agents and screened for plasmid DNA. All 65 isolates of 0 1 57

were resistant to four or more antimicrobial agent tested. All strains were resistant

to bacitracin ( 1 00%), methicillin ( 1 00%) and vancomycin ( 1 00%). All strains were

susceptible to cephalosporin, kanamycin, nalidixic acid and furazolidone. Fourty

isolates contained plasmid ranging in sizes from 1 .38 megadalton to 86.0

megadalton; and 29 possessed the 60.0 megadalton E. coli 0 1 57 serotype-specific

plasmid (pOl 57) of EHEC. Two of eight selected E. coli 0157 isolates were

observed to transfer their resistant phenotypes at frequencies 2.7 X 1 0-7 to 3 .0 X 1 0-6

per donor cells. The concomitant transfer of donor plasmids in two of the selected

E. coli 0 1 57 isolated was also detected. These results may suggest that antibiotic

resistance among the selected E. coli 0 1 57 isolates were encoded on conjugative R

plasmids.

xii

Abstrak tesis diserahkan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan untuk ljazah Master Sains.

PROFAIL PLASMID DAN KERENTANGAN ANTIBIOTIK ESCHERICHIA COLI DAN STRAIN-STRAIN ESCHERICHIA COLI 0157

Oleh

SAHILAH ABO. MUTALIB

MEl 1 997

Pengerusi : Dr. Son Radu.

Fakulti: Sains Makanan dan Bioteknologi.

Perhatian telah diberikan ke atas Enterohemorragik Escherichia coli

0 1 57:H7 kerana ia menyebabkan implikasi yang jarang berlaku seperti hemorrhagik

kolitis, hemolitik uremik sindrom dan thrombotik thrombositopenik purpura.

lnsiden yang disebabkan oleh E. coli 0 1 57 telah dilaporkan diseluruh dunia tetapi

kejadian jangkitan yang berlaku di Malaysia belum pernah diketahui. Dari Mac

sehingga lun 1 996, dua puluh lima sampel telah didapati dari 4 buah pasar-raya di

Selangor dan Wilayah Persekutuan Malaysia. Dua puluh lima gram sampel daging

telah dikayakan dalam 225 ml larutan EC yang mengandungi novobiocin (20 mg/I)

selama 4 jam pada 37°C dan diplatkan ke atas agar MacConkey. Sembilan belas

sehingga lima puluh koloni penapai laktosa per sampel (n=109 1 ) telah dipindabkan

di atas agar Eosin Metillin Biru, agar Sorbitol MacConkey dan agar Cefizim Telurit

Sorbitol MacConkey. Enam puluh lima dari 1 1 2 kilauan metalik dan koloni-koloni

sorbitol-negatif telah memberikan keputusan yang positif bila diujikan dengan kit

xiii

ujian agglutinasi latek antigen 0 1 57 yang dibekalkan dari Oxoid dan Serobact.

Sembilan belas (76%) sampel telah menunjukkan kehadiran positif E. coli 0 157. Ke

enam puluh lima strain E. coli 0 1 57 telah diuji kesensitifannya terhadap 20 agen

antimikrobial dan diskrinkan untuk DNA plasmid. Kesemua 65 asingan, rentang

kepada empat atau lebih agen mikrobial yang diujikan. Selain dari itu kesemua

enam puluh lima strain menunjukkan kerentangan terhadap bacitrasin, ( 1 00%),

meticillin ( 1 00%) dan vankomisin ( 1 00%). Bagaimanapun, kesemua strain sensitif

terhadap cephalosporin, kanamisin, acid nalidizik and furazolidon. Empat puluh

asingan mengandungi saiz DNA plasmid yang berjulat dari 82.0 megadalton

sehingga 1 .38 megadalton; dan dua puluh sembilan memiliki 60.0 megadalton

plasmid yang spesifik terhadap serotip (P01 57) EHEC. Dua dari 1 2 asingan E. coli

0 1 57 telah ditunjukkan memindahkan penotifkerentangan pada frekuensi 2 .7 X 1 0-7

to 3.0 X 1 0-6 per sel penderma. Pemindahan DNA plasmid juga didapati seiring

terhadap kedua-dua E. coli 0 1 57. Keputusan tersebut mencadangkan bahawa

kerentangan antibiotik di antara asingan E. coli 0 1 57 mungkin dienkodkan di atas

plasmid R konjugatif.

xiv

CHAPTER 1

INTRODUCTION

Escherichia coli was first described by Theobald Escherich in 1 885 (Sojka,

1 965). He examined the faeces of new-born breast-feeding babies and found that

they contained bacteria; he called this microorganism as Bacterium coli commune

which is now accepted as E. coli. It was first considered to be a harmless

saprophytes. However in 1 889, Laraeke (Sojka, 1 965) demonstrated that this

coliform bacteria existed in cases of appendicitis with peritonitis, which suggested

that it might be pathogenic.

The presence of E. coli in food is always associated with lack of cleanliness,

inadequate processing, and more specifically indicating the occurrence of fecal

contamination. Thus, the detection of E. coli can be used to asses the sanitary

quality of food, as well as in water and is considered primarily as an indicator

organism for the possible presence of pathogenic bacteria. E. coli also carries a

functional system for lactose utilisation study which led to the formulation of the

operon model for regulation of expression of prokaryotic genetic material (Glass,

1 982).

This microorganism has been extensively studied, and it is known that E. coli

commonly exist in the lower part of the intestine of most warm-blooded animals and

recognised as opportunistic pathogen because of its continuous presence in gut. It

2

has ample opportunity to cause extraintestinal diseases of any type when local or

general immunity has been compromised in favour of bacteria. Most common in

man are urinary tract infections, of which majority are cause by E. coli. Other

infections caused by E. coli are gall bladder, peritoneal cavity or wound infections.

In rare instances it may cause meningitis, septicemia, endocarditis and arthritis in

animal (Soltys, 1 979). In 1 982 two outbreaks of acute bloody diarrhea occurred in U.

S. A (State of Oregon and Michigan) (Riley et al. , 1 983) where E. coli was

recognised as the pathogen. The new strain was identified as E. coli 0 1 57:H7 which

is now associated with food-related outbreaks of an unusual gastrointestinal illness.

The illness is generally quite severe and can cause three different syndromes;

haemorrhagic colitis, hemolytic uremic syndrome and thrombotic thrombocytopenic

purpura.

Initially, infection due to E. coli 01 57:H7 was considered rare because of its

lack of frequent isolation prior to 1 982; however, subsequent experience has shown

that the organism can be isolated with such an appreciable frequency that it is now

not considered a rare serotype (Doyle, 1 99 1 ). E. coli 0 1 57 :H7 is now recognised as

an important cause of foodborne disease, with outbreaks reported in U. S. A.,

Canada and United Kingdom (Doyle, 1 99 1 ). Several additional outbreaks of E. coli

01 57:H7 infections have been reported in Mexico (Cravioto et al., 1 990), China (Xu

et al., 1 990), Argentina (Lopez et al., 1 989) and Belgium (Pierard et al., 1 990). The

illnesses caused by E. coli 0157:H7 appears to occur throughout much of the world

(Doyle, 1 99 1 ). However, no illness caused by E. coli 01 57:H7 has been reported yet

in Malaysia.

3

Most outbreaks due to E. coli 01 57:H7 have been associated with eating

undercooked ground beef or less frequently drinking raw milk. Surveys of retail raw

meats and poultry revealed that E. coli 01 57:H7 was detected in 1 .5 to 3.5% of

ground beef, pork, poultry and lamb. Dairy cattle, especially young animals, have

been identified as a reservoir (Doyle, 1 991). Faith e t al. ( 1 996) reported that the

range prevalence of E. coli 0157:H7 strains in herds of dairy and beef cattle was

1 .8% (1 9 of 1 068 herds) to 1 6% (4 of25 herds), respectively.

E. coli 01 57:H7 is typical of most E. coli, but does possess distinguishing

characteristics. One distinguishing feature of E. coli 0 1 57 :H7 is its lack of rapid

(less than 48 hours) fermentation of sorbitol. This has being used as the basis of the

most common screening procedure for this organism (Neill et al. , 1 993). E. coli

0 157:H7 does not possess B-glucuronidase activity and hence cannot hydrolyse 4-

methylumbelliferone glucuronide (MUG) to a fluorogenic product; it is therefore

MUG negative (Doyle and Schoeni, 1 984). The organism has no unusual heat

resistance; heating ground beef sufficiently to kill typical strains of Salmonellae will

also kill E. coli 0 1 57:H7. The mechanism of pathogenicity has not been fully

elucidated (Doyle, 1 99 1 ), but clinical isolates produce one or more verotoxins which

are believed to be important virulence factors.

Griffin et al. ( 1 988) reported that almost all strains of E. coli 0 1 57:H7 are

susceptible to ampicillin, trimethoprim-sulfamethoxazole, tetracycline and

quinolones, although they are resistant to erythromycin; metronidazole and

vancomycin. However, in the past few years, several reports had been published on

the emergence of multiply antibiotic resistance E. coli 0 1 57 :H7 (Swerdlow et

al. 1 992; Kim et al. , 1 994; and Farina et al., 1 996).

4

Since dairy cattle of young animals have been identified as a reservoir of E.

coli 01 57:H7 and the contamination of retail meats may probably occur during the

processing of meats at the retail level (Sekla et ai., 1 990), attempts are made to

isolate E. coli 0 1 57:H7 from imported frozen beef.

Objectives

The objectives of this study are:

1 . To isolate E. coli and E. coli 01 57:H7 from imported frozen beef and its

biochemical tests.

2. To determine antibiotic susceptibility and plasmid profiles of the E. coli and E.

coli 01 57:H7.

3. To carry out genetics transfer study on selected isolates. Such study would

provide information on the possible correlation between the presence of plasmids

and antibiotics resistance.

CHAPTER 2

LITERATURE REVIEW

Escherichia coli

Taxonomy

Escherichia coli is classified in the family Enterobacteriaceae and its

taxonomic features includes Gram-negative, asporogenic, straight rods that may be

peritrichously flagellated or nonmotile (Orskov, 1 984). It coagulated milk, producing

acid and gas. The ability to ferment certain carbohydrates, producing acid and gas

was soon adapted as a basis for the differentiation of closely related enteric bacteria

such as Shigella and Salmonella.

Five species of Escherichia now are recognised; E. coli, E. herman ii, E.

blattae, E. vulneris and E. fergusonii (Edwards and Ewing, 1 986). These were

delineated by means of DNA relatedness studies by Brenner and Brenner et al.

(Cited by Edwards and Ewing, 1 986). E. hermanii was differentiated earlier and was

called a biogroup of E. coli followed by E. blattae. The DNAs from all above

mentioned species of Escherichia are related to those of E: coli (average, 84%)

(Edwards and Ewing, 1 986).

5

6

Generally, the genus of Escherichia is composed of motile or non-motile bacteria

that conform to the definitions of the family ENTEROBACTERIACEAE and the tribe

ESCHERICHIEAE. Both acid and gas are formed from a wide variety of

fermentable carbohydrate, but aerogenic biotypes occur; salicin is fermented by

many cultures but inositol is not utilised and adonitol is utilised by members of only

one species. Lactose often is fermented rapidly, some strains utilise it slowly, and

some fail to ferment this substrate. Lysine and ornithine are decarboxylated by the

majority of cultures, acid is formed from sodium mucate, and sodium acetate

frequently is utilised as a sole source of carbon.

E. coli, the type species in this family was fully reviewed by Glass ( 1 982) as

a favourite organism for all types of microbiological studies. Prominent in any list

of the advantages of research on bacteria must be the rapid growth and limited

nutritional requirements of those microorganism.

Genome size (including chromosomal and extrachromosomal DNA) in E.

coli strain varies from 2.3 X 1 09 to 3 .0 X 1 09 daltons. The G + C content ranges

from 49 to 52% (Selander et aI. , 1 987).

Habitat

Most strains of E. coli are harmless commensal members of the intestinal

flora of mammals and, to an undetermined extent, birds in which some strain adhere

to the intestinal mucous while others are only temporary transients in the lumen of

the colon. E. coli is the major anaerobe of the large intestine, occurring in the

7

intestine, in normal densities of about 1 06 cells per g of colon contents. It is a minor

component of the total intestinal flora, which consists largely of obligate anaerobes

and in the aggregate reaches 1 01 1 cells per g of colon contents (Selander et al. ,

1 987).

E. coli is also one of the first of the intestinal bacteria to colonise the

newborn, generally being derived from the faeces of the mother in both humans and

animals (Baltelheim et al., 1 974) and may be present in the cecum and lower

intestine of mammals within hours or day after birth.

Growth in the intestine is much slower than m laboratory culture, the

doubling time being estimated at about 1 day. While most strains of E. coli live as

benign commensals, many perhaps all, are opportunistic pathogens of humans and

other animals (Soltys, 1 979). In 1 982, two outbreaks of acute bloody diarrhea

occurred in the state of Oregon and Michigan where E. coli (E. coli 0 1 57 :H7) was

first recognised as a pathogen (Riley et aI. , 1 983).

E. coli is also a prime agents in surgical and other nosocomial (hospital-

acquired) infections (wound, secondary pneumonia, peritonitis) in compromised

patients (Soltys, 1 979). In addition E. coli is the major cause of neonatal septicemia,

neonatal meningitis and urinary tract infection in humans and of a variety of invasive

disease in mammals and birds (Sojka, 1 965), including mastitis in cattle and sepsis

in chicken (Sojka, 1 965). It is also a leading cause of diarrheal disease in humans

and other animals.

8

The intestine and tissues of warm blooded animals are the primary habitats of

E. coli, secondary habitats are soil, sediment and water (Caugant, 1 983). Under

extreme conditions, such as around cattle feed lots, densities may reach fecal levels

in the soil and 1 04 cells per ml in polluted water. In surface soils underlying manure,

E. coli is reported to survive for several months, but normally it may live in soil or

water for only a few days without dividing (Selander et aI., 1 987). Human and food

animals should perhaps also be regarded as a secondary environment of E. coli.

Isolation and Identification

E. coli can be easily isolated by growmg the suspensIOn samples on

MacConkey or eosin methylene blue agar (EMBA). The colonies growing on

MacConkey medium are rose-coloured owing to the action of acid produced during

fermentation of lactose. However, most primary isolation of E. coli rely on EMBA.

This medium is lactose-peptone agar containing eosin methylene blue. The colonies

growing on this media will have a greenish metallic sheen due to the reflection of

light. Therefore, E. coli can be distinguished from Aerobacter aerogene and

Candida albicans which also have the ability to grow on the same media.

Descriptions of media and reagents used for E. coli and other

Enterobacteriaceae can be found in several common laboratory manuals, for

example Edwards and Ewing ( 1 986). To identify the presumptive E. coli, they

(Edwards and Ewing, 1 986) suggested a list of biochemical test as shown in

Table 1 .

9

Table 1 : The biochemical reactions of Escherichia coli.

Test or substrate

Hydrogen sulphide (TSI agar) Urease Indole Methyl red Voges-Proskauer Citrate (Simmon's) KCN Motility

Gelatin (22°C) Lysin decarboxylase Arginine dihydrolase Ornithine decarboxy lase Phenylalanine deaminase Glucose

acid gas

Lactose Sucrose

Mannitol Dulcitol Salicin Adonitol Inositol

Sorbitol Arabinose Raffinose

Key + (+)

+ or-- or + + or (+) d F TSI a

E. coli Test or substrate E. coli Sign %+(%+) Sign %+(%+)-- o· Rhamnose d 83.5 (3.4)

- o· Malonate - 0 + 96.3 Mucate + 91.6 + 99.9 Christensen's citrate d 18.2 (22.6) - 0 Jordan's D-tartrate + 97.6 - 0.2 (0.3) Pectate - 0 - 0 Sodium acetate + or (+) 83.8 (9.7) + or (+) 62.1 Ammonium salts + 94.5 (1.7)

-

d d

d

-

+ + + d

+ d d -

-

d + d

glucose agar 0 Sodium alginate - 0 80.6 ( 1 .5) Lipase 16.3 (39.1) Com oil - 0

57.8 (8) Tributyrin - 0

0 Maltose + 90.6 (2.4)

Xylose d 82.8 (6.6) 100 Trehalose + 98.2 (1.8) 92 Cellobiose - 1 (3) 91.6 (4.2) Glycerol + or (+) 89 (8.3) 53.7 (5.5) Alpha- - 0

methylglucoside 97.5 Erythritol - 0 49.3 ( 1 8) Esculin d 30.9 (19.7) 36 (12.3) Beta-galactosidase + >96 1 Nitrate to nitrite + 99.8 (0.2) 0.9 (0.2) Oxidation- F 100

fermentation 80.3 (I) Oxidase - 0 99.3 (0.5) DNase (22°C) - 0 49.4 (2. 1 ) Yellow pigment - <0.1

90% or more positive within 1 or 2 days. positive reaction after 3 or more days (decarboxylase tests: 3 or 4 days). no reaction (90% or more) in 30 days. most cultures positive; some strains negative. most strains negative; some cultures positive. most reactions occur within I or 2 days; some are delayed. different reactions, +., (+), -. Fermentative. Triple sugar iron.

An occasional strain may produce abundant hydrogen sulfide and an occasional culture may hydrolyse urea. Both of these reactions

are mediated by (different plasmid). The urease reactions are weak and usually are not apparent until after 24 hours of incubation.

(Source: Edwards and Ewing, 1 986).

10

Serological Classification

Serological classification of E. coli is based on three kinds of antigens that

occur in E. coli. The serotyping scheme was developed by Khaufman in 1 947

(Doyle and Padhye, 1 989) and divided into three categories:

a. Thermostable somatic "0" antigens closely resembling those of the Salmonella.

b. H or flagella antigens.

c. K or surface antigens.

The 0 antigen, which is based on the antigenicity of O-specific

polysaccharide of the cells outer membrane lipopolysaccharides, is a thermo stable

(stable at 1 00°C for 2 hours) surface antigen found in all smooth (S forms)

Enterobacteriaceae (Doyle and Padhye, 1989). The 0 antigen forms the basis for

classifying E. coli into serogroups, of which there are more than 1 70 (Orskov, 1 984).

Within each serogroup, are one or more serotypes that are based on the H antigen.

For example 0126:H27 and 0126:K represent two serotypes of E. coli within the

same serogroup. H antigen are heat-labile flagella antigens compose of protein.

Three varieties of K antigen have been described: the heat-labile L antigen;

the heat-stable A antigen which is associated with capsule formation and the B

antigen which is partially altered by heat (Soltys, 1 979). Table 2 gives further

information about K antigen.